The aforementioned selective plane illumination microscopy (SPIM) technique underlying the present invention is described, for example, in Lindek et al; Journal of modern optics, 1999, vol. 46, no. 5, 843-858.
A microscope which operates according to the SPIM method is described in DE 102 57 423 A1. In this microscope, a sample is illuminated by a thin light strip, while the viewing takes place perpendicularly to the plane of the illuminating light strip. Here, illumination and detection are carried out via two separate optical trains having separate optics, in particular, two separate objectives oriented perpendicular to each other. The light strip is produced by the illumination objective and an upstream cylindrical optical element. For image acquisition, the sample is moved through the light strip, which is stationary relative to the detector, to capture fluorescent and/or scattered light layer by layer using an area detector. The tomographic image data so obtained can then be assembled into one data set representative of a three-dimensional image of the sample. In order to produce as thin a light strip as possible, the illumination objective must have a correspondingly high numerical aperture. Moreover, the free working distance of the illumination objective must be large enough to prevent collision with the observation objective. The perpendicular arrangement of the two objectives can be disadvantageous in the imaging of certain samples, especially biological ones. For example, it is often not possible to place spherical objects in a collision-free manner under a right-angled arrangement of objectives. In addition to the extreme requirements imposed on the sample preparation, frequently unwanted shading occurs in the sample.
In a modified SPIM technique described in WO 2010/012980 A1, illumination and detection are performed using the same objective. To this end, the entrance pupil of the objective is under-illuminated at an off-center position; i.e., the illumination beam passes through a portion of the entrance pupil that is transversely offset from the optical axis. A cylindrical lens upstream of the objective produces a light sheet in the sample, which light sheet is inclined to the optical axis of the objective. The sample region illuminated by this light sheet is then imaged by the objective onto a detector. However, this device is designed exclusively for oblique illumination of the sample by means of a light sheet and does not allow for a different use, and especially not for point-by-point confocal scanning of the sample or variation of the spatial light intensity distribution of the light sheet, and in particular, not for illumination by a light strip oriented perpendicular to the optical axis of the objective.
German Patent Publication DE 10 2004 034 957 A1 describes an arrangement for microscopic observation through a microscope objective, where light guides for the light illuminating the sample are provided in the objective housing outside the lens optics. The illuminating light initially propagates parallel to the optical axis of the objective within the light guide, and then strikes reflectors of small aperture, which are mounted on the objective housing and which, with the aid of additional imaging elements, focus the illuminating light into the sample in a direction perpendicular to the optical axis of the microscope objective, and thus perpendicular to the viewing direction. Here, too, planar illumination of the sample is provided according to the SPIM principle. Although the use of a microscope objective configured in this manner does indeed eliminate the need to use an additional objective for the illuminating light, the special design of this special objective with additional light guides and reflectors is technically very complex and expensive.